Reduction of intracellular pH (pHi) by acute reduction of extracellular pH (PhE) leads to hyperthermia sensitization and sensitization to certain chemotherapeutic agents. The global hypothesis of this program is that acute acidification provides a mechanisms by which melanoma can be sensitized by chemical means to pH-dependent therapeutic agents such as hyperthermia. We have shown that hyperglycemia alone is unlikely to provide significant sensitization to hyperthermia. Strategies have been developed to enhance melanoma to hyperthermia by acute tumor acidification. This research program will provide new mechanistic insights into: The effects of acidification alone or combined with hyperthermia on DNA replication in a defined molecular system (Project 1); The regulation of pHi and methods to inhibit proton transport in melanoma cells adapted at growth at low pHe (Project 2); The cytoskeletal resistance and upregulation of hsp27, hsp60 and hsp72 that accompany growth of melanoma cells at low pHe (Project 3); The chemical methods to enhance hyperglycemia-induced acidification in human melanoma xenografts (Project 4) and The sensitization by acute acidification of the response of melanoma xenografts to hyperthermia relative to normal tissues (Project 4). A subcontract to the University of Pennsylvania will measure xenograft pHi and other metabolic parameters by non-invasive magnetic resonance techniques. This research plan is an outgrowth of the progress made with previous N.C.I. support. Four interactive projects and two cores are proposed. Projects 1 & 3 provide molecular and cellular mechanisms of effects of acidification on response to hyperthermia. Project 2 seeks to understand regulation of pHi and identify novel drugs to inhibit proton pumping mechanisms to induce intracellular acidification. Project 2 measures pHi for all projects. Project 4 validates the molecular and cellular findings in vivo by determining pHe, pHi, pO2, blood flow and glucose metabolites in xenografts and by comparing the response of xenografts and normal tissue to hyperthermia. Furthermore, Project 4 provides melanoma cells and response of xenografts and normal tissue to hyperthermia. Furthermore, Project 4 provides melanoma cells and tissues to Projects 1, 2 & 3 to test the hypotheses proposed in vitro. The Administrative Core provides administrative support, statistical & data management and external review for all projects. The Animal Care Core purchases and provides care for animals for xenografts to provide melanoma cells and tumors for all projects.